Original Article http://dx.doi.org/10.5115/acb.2015.48.4.258 pISSN 2093-3665 eISSN 2093-3673

Haversian system of compact bone and comparison between endosteal and periosteal sides using three-dimensional reconstruction in rat

Jeong-Nam Kim1, Jun-Young Lee1, Kang-Jae Shin2, Young-Chul Gil2, Ki-Seok Koh2, Wu-Chul Song2 1Department of Biomedical Laboratory Science, Masan University, Masan, 2Department of Anatomy, Research Institute of Medical Science, Konkuk University School of Medicine, Seoul, Korea

Abstract: The current model of compact bone is that of a system of Haversian (longitudinal) canals connected by Volkmann’s (transverse) canals. Models based on either histology or microcomputed tomography do not accurately represent the morphologic detail and microstructure of this system, especially that of the canal networks and their spatial relationships. The aim of the present study was to demonstrate the morphologic pattern and network of the Haversian system and to compare endosteal and periosteal sides in rats using three-dimensional (3D) reconstruction. Ten Sprague-Dawley rats aged 8–10 weeks were used. The femurs were harvested from each rat and fixed, decalcified with 10% EDTA-2Na, serially sectioned at a thickness of 5 mm, and then stained with hematoxylin and eosin. The serial sections were reconstructed three-dimensionally using Reconstruct software. The Haversian canals in the endosteal region were found to be large, highly interconnected, irregular, and close to neighboring canals. In contrast, the canals in the periosteal region were straight and small. This combined application of 3D reconstruction and histology examinations to the Haversian system has confirmed its microstructure, showing a branched network pattern on the endosteal side but not on the periosteal side.

Key words: Haversian system, Compact bone, Three-dimensional reconstruction, Microstructure, Histology, Rat

Received January 9, 2015; Revised September 11, 2015; Accepted October 1, 2015

Introduction Terminologia Histologica [1-3]. The canals have a concentric lamellar organization and are of equal size. The bone is vas­ The morphological and functional unit of the bone is the cularized by vessels that penetrate the matrix from the perio­ Haversian system, with textbooks diagrams showing Haver­ steum. sian or central (longitudinal) canals connected by Volkmann’s The microstructure of the compact bone is important to or perforating (transverse) canals. Officially, Haversian and bone remodeling and modification in pathologic conditions. Volkmann’s canals are “nutrient and perforating canal” in Therefore, knowledge of the microstructure of compact bone such as the Haversian system is important to understanding remodeling activity, distribution analysis, the pathogenesis of the osteoporosis, and other mechanisms. However, many Corresponding author: Wu-Chul Song studies of the Haversian system have focused only on the Department of Anatomy, Konkuk University School of Medicine, 120 canals themselves due to methodology limitations asso­ Neungdong-ro, Gwangjin-gu, Seoul 05029, Korea Tel: +82-2-2030-7819, Fax: +82-2-2030-7845, E-mail: [email protected] ciated with cross-sectional histology and microcomputed tomography [4-7], rather than on the general canal system Copyright © 2015. Anatomy & Cell Biology

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Morphological characteristics of the Haversian system Anat Cell Biol 2015;48:258-261 259 or the spatial relationships between the two types of canals. Tissue processing Knowledge of the overall composition of the Haversian sys­ After dehydration and infiltration, the paraffin-embedded tem as well as the constituent canals themselves is very impor­ specimens were cut transversely into 150 to 200 serial sections tant when investigating changes relative to the normal system. at a thickness of 5 mm for 3D reconstruction. The sectioned The aim of the present study was to demonstrate the ac­ tissues were then stained with hematoxylin and eosin. tual microstructure and morphologic pattern of the Haver­ sian system of compact bone using histology and three- 3D reconstruction dimensional (3D) reconstruction, rather than relying on All of the stained sections were photographed using a virtual images. This described reconstruction technique can provide a general view of the architecture of the Haversian sys­tem and further the understanding of pathologic progres­ Marrow cavity sion of the compact bone.

Materials and Methods

Materials and sampling Twenty Sprague-Dawley rats aged 8–10 weeks were used. The animals were perfused with 150 ml of saline followed by 600 ml of fixative containing 4% paraformaldehyde in phosphate-buffered saline. The femur was harvested and de­ calcified in 5% EDTA-2Na. The specimens were trimmed to 3 mm to make them easier to handle and to facilitate the acquisition of images during fixation. Each specimen was ob­ tained from the anterior midshaft of the femur. Fig. 1. Transverse section of the rat femur stained with hematoxylin and eosin. Scale bar=100 mm.

Case 1 Case 2 Case 3

Superior view

a)

Endosteal oblique view

Fig. 2. Three-dimensional reconstruc­ a) tion of the Haversian system. Different colors represent the different networks

a) of the Haversian and Volkmann’s canals. Transparent gray shows the outline of the bone fragment. Many, large, Periosteal and complex reddish networks of the oblique Haversian and Volkmann’s canals are view located on the endosteal side, while rela­ tively simple networks are located on the periosteal side. All images have the same magnification. a)Endosteal side.

www.acbjournal.org http://dx.doi.org/10.5115/acb.2015.48.4.258 260 Anat Cell Biol 2015;48:258-261 Jeong-Nam Kim, et al

2,048×1,536-pixel digital CCD camera (DP70, Olympus, in the Haversian system indicate that the central canal size Tokyo, Japan). The serial images were aligned manually using is equal in the endosteal and periosteal regions of compact computer software, the Haversian and Volkmann’s canals were bone [1-3]. However, we found that the Haversian system has segmented manually, and all structures were reconstructed a complex pattern of organization that is dominated by bran­ three-dimensionally. “Reconstruct” software was used to pro­ ching or Volkmann’s canals. Our method of combining 3D duce the 3D images of the Haversian system (this software reconstructions with histology examinations has confirmed can be downloaded from http://synapses.clm.utexas.edu/ the actual morphologic pattern of the Haversian system. tools/reconstruct/reconstruct.stm) [8]. The main result of the present study is that the Haversian system is convoluted and closely interconnected in the endo­ Results steal region, while being straight in the periosteal region. The canals are significantly larger in the endosteal region than in The Haversian canals were arranged into concentric rings the periosteal region. Moreover, there are more interstitial and connected to each other by oblique perforating canals in lamellae in the periosteal region. the transverse sections (Fig. 1). However, the network pattern, The microstructure of the Haversian system is charac­ spatial relationship between Haversian and Volkmann’s terized by a branched network pattern that differs between canals, and difference between endosteal and periosteal sides the endosteal and periosteal sides. Because previous studies of were difficult to identify in the transverse histology sec­ the pathology of the compact bone during osteoporosis have tions. In contrast, all of these features were clearly evident generally focused on the thickness of the compact bone, the in the reconstructed 3D models (Fig. 2). The basic structure present results are useful in providing a general view of the comprises Haversian canals that are large in the endosteal architecture of the Haversian system and for understanding region and highly interconnected. The canals in the endosteal pathologic progression of the compact bone. In particular, region appear irregular and close to neighboring canals. In the in cases of osteoporosis the thickness of the cortical bone is other hand, the canals of the periosteal region are relatively reduced even though the external diameter remains almost small and less interconnected; that is, the network density is intact. It might be that the degree of bone resorption is much significantly higher in the endosteal region than in the perio­ greater in the endosteal region due to the presence of a large steal region. The Haversian canals are straight and long and and highly interconnected Haversian system there. parallel to the length of femur. The Volkmann’s canals run The present results have demonstrated that the network perpendicular to the Haversian canals, interconnecting the of the Haversian system can be revealed only by combining latter with each other and the periosteum, and extending in 3D reconstruction with histology observations. We therefore random directions at various angles. further suggest that 3D reconstruction could also be a highly effective tool in other areas of morphology research. Discussion Acknowledgements Haversian canals are a series of tubes around narrow channels formed by lamellae. The Haversian canals sur­ This work was supported by the Korea Research Founda­ round blood vessels and nerve fibers throughout the bone tion Grant funded by the Korean Government (MOEHRD, and communicate with osteocytes. The canals and the sur­ Basic Research Promotion Fund)(313-2008-2-E00005). rounding lamellae are called a Haversian system (or an osteon). A Haversian canal generally contains one or two References capillaries and nerve fibers. The spaces between Haversian systems contain interstitial lamellae. The osteonal pattern 1. Kessel RG, Kardon RH. Tissues and organs: a text-atlas of of compact bone is gradually built around the intracortical scanning microscopy. New York: W.H. Freeman and Co.; 1979. 2. Ovalle WK, Nahirney PC. Netter’s essential histology. Phila­ vessels by the progression of the cutting cones in a process of delphia: Elsevier; 2008. p.146. secondary remodeling; therefore, the central canal size can be 3. Mescher AL. Junqueira’s basic histology. 13th ed. New York: used as an index of the remodeling activity [9]. McGrawHill; 2013. p.145-8. Textbook diagrams of the Haversian and Volkmann’s canals 4. Tanaka M, Yamashita E, Anwar RB, Yamada K, Ohshima H,

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Nomura S, Ejiri S. Radiological and histologic studies of the 7. Cooper DM, Matyas JR, Katzenberg MA, Hallgrimsson B. mandibular cortex of ovariectomized monkeys. Oral Surg Oral Comparison of microcomputed tomographic and microradio­ Med Oral Pathol Oral Radiol Endod 2011;111:372-80. graphic measurements of cortical bone porosity. Calcif Tissue 5. Particelli F, Mecozzi L, Beraudi A, Montesi M, Baruffaldi F, Int 2004;74:437-47. Viceconti M. A comparison between micro-CT and histology for 8. Fiala JC. Reconstruct: a free editor for serial section microscopy. the evaluation of cortical bone: effect of polymethylmethacrylate J Microsc 2005;218(Pt 1):52-61. embedding on structural parameters. J Microsc 2012;245:302-10. 9. Pazzaglia UE, Zarattini G, Giacomini D, Rodella L, Menti AM, 6. Britz HM, Jokihaara J, Leppänen OV, Järvinen T, Cooper DM. Feltrin G. Morphometric analysis of the canal system of cortical 3D visualization and quantification of rat cortical bone porosity bone: An experimental study in the rabbit femur carried out using a desktop micro-CT system: a case study in the tibia. J with standard histology and micro-CT. Anat Histol Embryol Microsc 2010;240:32-7. 2010;39:17-26.

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